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In a field survey in the Taiwan Strait during April 2016, the species composition and the domoic acid production of the diatom genus Pseudo-nitzschia were investigated. A total of 80 strains of Pseudo-nitzschia were established, and species identification was determined based on a combination of morphological and molecular data. Fourteen taxa were recognized, i.e., P. americana, P. brasiliana, P. calliantha, P. cuspidata, P. galaxiae, P. lundholmiae, P. multiseries, P. multistriata, P. pseudodelicatissima, P. pungens var. aveirensis, P. pungenus var. pungens and P. sabit, as well as two novel species P. chiniana C.X. Huang & Yang Li and P. qiana C.X. Huang & Yang Li. Morphologically, P. chiniana is characterized by striae comprising one or two rows of poroids, and valve ends that are normally dominated by two rows of poroids within each stria. Whereas P. qiana is unique by having a narrow valve width (1.3–1.5 μm) and sharply pointed valve ends. Both taxa constitute their own monophyletic lineage in the phylogenetic analyses inferred from LSU and ITS2 rDNA, and are well differentiated from other Pseudo-nitzschia species. Pseudo-nitzschia chiniana forms a group with P. abrensis and P. batesiana in LSU and ITS trees, whereas P. qiana is sister to P. lineola. When comparing ITS2 secondary structure, five CBCs and seven HCBCs are recognized between P. chiniana and P. abrensis, and four CBCs and ten HCBCs between P. chiniana and P. batesiana. Two CBCs and eight HCBCs are found between P. qiana with P. lineola. The ability of the strains to produce domoic acid was assessed, including a potential toxin induction by the presence of brine shrimps. Results revealed production of domoic acid in six strains belonging to three species. Without presence of brine shrimps, cellular DA (pDA) was detected in four P. multiseries strains (1.6 ± 0.3, 26.6 ± 2.7, 68.3 ± 4.2 and 56.9 ± 4.7 fg cell−1, separately), one strain of P. pseudodelicatissima (0.8 ± 0.2 fg cell−1) and one strain of P. lundholmiae (2.5 ± 0.4 fg cell−1). In the presence of brine shrimps, pDA contents increased significantly (p

The globally distributed Chaetoceros elegans belongs to the Chaetoceros lorenzianus (C. lorenzianus) complex and is characterized by having tear-shaped setae poroids. Several strains of C. elegans were established from Chinese coastal waters. The vegetative cells and the resting spores were observed using light and electron microscopy. Phylogenetic analyses of two nuclear ribosomal RNA genes (SSU and the D1–D3 region of LSU) and the internal transcribed spacer (ITS) revealed that the C. elegans strains clustered into three clades, corresponding to different morphotypes. Based on the type material, the delineation of C. elegans was amended, and two new taxa, (Chaetoceros macroelegans) C. macroelegans sp. nov. and (Chaetoceros densoelegans) C.densoelegans sp. nov., were described. The two new taxa are featured by the presence of two types of setae poroids, tear-shaped and round-oval setae poroids, whereas only tear-shaped setae poroids are seen in C. elegans. The setae base is distinct in C. elegans, but absent or short in the two new taxa. In C. macroelegans, the tear-shaped poroids on the intercalary setae are larger and less densely spaced than in the other two species. The round-oval setae poroids are more densely spaced in C.densoelegans than in C. macroelegans, although they have more or less the same size. Resting spores characterize the two new taxa, but are unknown in the amended C. elegans. When comparing the ITS2 secondary structure, two and four compensatory base changes (CBCs) distinguish C. elegans from C. macroelegans and C.densoelegans, respectively. Between the two new taxa, no CBC but five hemi-CBCs (HCBCs) are present. The shape, size and density of the setae poroids, as well as the morphology of the resting spores, are important characteristics for species identification among the presently nine known species within the C. lorenzianus complex.

In March 2018, an algal bloom of Pseudo-nitzschia was detected, for the first time, in a semi-enclosed lagoon in Miri, Sarawak, Malaysia Borneo. The plankton samples were collected for cell enumeration and species identification by electron microscopy and molecular characterization. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was performed to detect and quantify the neurotoxin domoic acid (DA) in both the plankton and shellfish samples. The abundance of Pseudo-nitzschia cells ranged from 5.6 × 105 to 3.5 × 106 cell L-1 during the bloom event. Morphological observation of the cells by transmission electron microscopy showed that the plankton samples comprised a single Pseudo-nitzschia morphotype resembling P. cuspidata. The ITS2 sequence-structure phylogenetic inference further supported the species identity as Pseudo-nitzschia cuspidata. Low levels of DA were detected in the plankton samples, with cellular DA, particulate DA, and dissolved DA of 257–504 fg DA cell-1, 676 ng L-1, and 15 ng L-1, respectively. The amount of DA, 8 μg g-1 tissue, was found present in the shellfish sample (Magallana sp.) which is below the regulatory limit of 20 μg DA g-1 tissue. The study documented, for the first time, DA contamination in shellfish that associated with bloom of P. cuspidata in the Western Pacific region.

To explore the species diversity and toxin profile of Pseudo-nitzschia, monoclonal strains were established from Chinese southeast coastal waters. The morphology was examined under light and transmission electron microscopy. The internal transcribed spacer region of ribosomal DNA was sequenced for phylogenetic analyses, and the secondary structure of ITS2 was predicted and compared among allied taxa. A combination of morphological and molecular data showed the presence of two new species, Pseudo-nitzschia hainanensis sp. nov. and Pseudo-nitzschia taiwanensis sp. nov. Pseudo-nitzschia hainanensis was characterized by a dumpy-lanceolate valve with slightly blunt apices and a central nodule, as well as striae comprising two rows of poroids. Pseudo-nitzschia taiwanensis was characterized by a slender-lanceolate valve, and striae comprising one row of split poroids. The poroid structure mainly comprised two sectors. Both taxa constituted their own monophyletic lineage in the phylogenetic analyses inferred from ITS2 rDNA and were well differentiated from other Pseudo-nitzschia species. Morphologically, P. hainanensis and P. taiwanensis could be assigned to the Pseudo-nitzschia delicatissima and the Pseudo-nitzschia pseudodelicatissima complex, respectively. Particulate domoic acid was measured using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), but no detectable pDA was found. With the description of the two new species, the species diversity of genus Pseudo-nitzschia reaches 58 worldwide, among which 31 have been recorded from Chinese coastal waters.

The diatom genus Pseudo-nitzschia, which has been associated with amnesic shellfish poisoning events globally, is also one of the key harmful microalga groups in Guangdong coastal waters, off the north coast of the South China Sea. In order to explore the diversity and toxigenic characteristics, Pseudo-nitzschia isolates were established. Based on a combination of morphological and molecular features, in total 26 different Pseudo-nitzschia taxa were identified, including two new species, P. uniseriata H.C. Dong & Yang Li and P. yuensis H.C. Dong & Yang Li. Morphologically, P. uniseriata is unique by having striae mainly comprising one row of poroids, which are simple without divided hymen internally, and each poroid containing one, seldom two sectors. Pseudo-nitzschia yuensis is characterized by having striae comprising one to two rows of poroids. In biseriate striae, the poroids are polygonal and irregularly distributed, and a discontinuous row of poroids may be present in the middle. In uniseriate striae, the poroids usually contain 1–5 sectors. Both taxa are well differentiated from other Pseudo-nitzschia species in phylogenetic analyses inferred from ITS2 sequence-structure information. Pseudo-nitzschia uniseriata is sister to P. lineola, whereas P. yuensis forms a group together with P. micropora and P. delicatissima. When comparing ITS2 secondary structure, two hemi-compensatory base change (HCBCs) are found between P. uniseriata and P. lineola. One compensatory base change (CBC) and four HCBCs are found between P. yuensis and P. delicatissima, and there is one CBC and five HCBCs between P. yuensis and P. micropora. The ability of cultured strains to produce particulate DA (pDA) revealed production of pDA in twenty-nine strains belonging to seven species: P. bipertita, P. caciantha, P. cuspidata, P. fraudulenta, P. fukuyoi, P. lundholmiae and P. multiseries. This is the first report of P. bipertita being toxic, with pDA content of 15.6 ± 2.1 fg cell–1. The presence of brine shrimps significantly increased pDA content in P. cuspidata, P. fukuyoi, P. lundholmiae and P. multiseries 1.4 to 7 times, and induced pDA production in P. fraudulenta from below detection limit to 17.5 ± 1.6 fg cell–1. The highest pDA concentration, 4830.5 ± 120.3 fg cell–1, was detected in P. multiseries, a level much lower than previous reports on P. multiseries from North America and Europe. Overall, the cellular toxin levels in Pseudo-nitzschia spp. were low in Guangdong coastal isolates.

The genus Pseudo-nitzschia has attracted attention because of production of the toxin, domoic acid (DA), causing Amnesic Shellfish Poisoning (ASP). Pseudo-nitzschia blooms occur frequently in Chinese coastal waters, and DA has been detected in several marine organisms, but so far no Pseudo-nitzschia strains from Chinese waters have been shown to produce DA. In this study, monoclonal Pseudo-nitzschia strains were established from Chinese coastal waters and examined using light microscopy, electron microscopy and molecular markers. Five strains, sharing distinct morphological and molecular features differentiating them from other Pseudo-nitzschia species, represent a new species, Pseudo-nitzschia simulans sp. nov. Morphologically, the taxon belongs to the P. pseudodelicatissima group, cells possessing a central nodule and each stria comprising one row of poroids. The new species is characterized by the poroid structure, which typically comprises two sectors, each sector located near opposite margins of the poroid. The production of DA was examined by liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses of cells in stationary growth phase. Domoic acid was detected in one of the five strains, with concentrations around 1.05-1.54 fg cell-1. This is the first toxigenic diatom species reported from Chinese waters.

Pseudo-nitzschia nanaoensis sp. nov. is described from waters around Nan'ao Island (South China Sea), using morphological data and molecular evidence. This species is morphologically most similar to P. brasiliana, but differs by a denser arrangement of fibulae, interstriae, and poroids, as well as by the structure of the valvocopula and the narrow second band. Pseudo-nitzschia nanaoensis constitutes a monophyletic lineage and is well differentiated from other species on the LSU and ITS2 sequence-structure trees. Pseudo-nitzschia nanaoensis makes up the basal node on the LSU tree, and forms a sister clade with a group of P. pungens and P. multiseries on the ITS2 tree. The ability of cultured strains to produce domoic acid was assessed, including its possible induction by the presence of a copepod and brine shrimp, by liquid chromatography-tandem mass spectrometry. However, no strains showed detectable domoic acid.

Analyses of the mitochondrial cox1, the nuclear-encoded large subunit (LSU), and the internal transcribed spacer 2 (ITS2) RNA coding region of Pseudo-nitzschia revealed that the P. pseudodelicatissima complex can be phylogenetically grouped into three distinct clades (Groups I–III), while the P. delicatissima complex forms another distinct clade (Group IV) in both the LSU and ITS2 phylogenetic trees. It was elucidated that comprehensive taxon sampling (sampling of sequences), selection of appropriate target genes and outgroup, and alignment strategies influenced the phylogenetic accuracy. Based on the genetic divergence, ITS2 resulted in the most resolved trees, followed by cox1 and LSU. The morphological characters available for Pseudo-nitzschia, although limited in number, were overall in agreement with the phylogenies when mapped onto the ITS2 tree. Information on the presence/absence of a central nodule, number of rows of poroids in each stria, and of sectors dividing the poroids mapped onto the ITS2 tree revealed the evolution of the recently diverged species. The morphologically based species complexes showed evolutionary relevance in agreement with molecular phylogeny inferred from ITS2 sequence–structure data. The data set of the hypervariable region of ITS2 improved the phylogenetic inference compared to the cox1 and LSU data sets. The taxonomic status of P. cuspidata and P. pseudodelicatissima requires further elucidation. © 2018 Phycological Society of America

A B S T R A C T Pseudo-nitzschia pungens is a planktonic marine diatom known to be widespread in tropical and temperate coastal waters. We examined the population genetic structure of tropical Southeast Asian populations of P. pungens and compared it with those of northern and southern temperate populations. The secondary structures of the nuclear encoded internal transcribed spacer (ITS) region of 164 strains of P. pungens were modeled and analyzed. The tree revealed three ITS entities: clade I (comprised of P. pungens var. pungens) was distributed mainly in northern temperate waters; clade II (comprised of both P. pungens var. pungens and var. cingulata) was mainly from the NE Pacific; and clade III (comprised of both P. pungens var. pungens and var. aveirensis) was restricted to tropical and warm-temperate waters. Hybrids of both P. pungens var. pungens and var. cingulata co-occurred in clades I and II. Sixty haplotypes were revealed from the sequences of 164 strains. Haplotype diversity inferred from the median-joining network was in accordance with phylogenetic analysis, further supporting the grouping of the P. pungens haplogroups. Our results revealed limited gene flow between P. pungens from tropical and temperate waters, and significant population structure, as estimated by an analysis of molecular variance (AMOVA), with 75% of the total ITS variation found among populations (KST = 0.75). This study suggests that distinct environmental clines, such as ocean thermohaline circulation, have a potential for fragmenting and dispersing global populations of P. pungens. Formation of the Isthmus of Panama, in particular, is speculated to play a role in this allopatric differentiation in P. pungens populations worldwide.

To clarify the genetic diversity of a potentially toxic pennate diatom, Pseudo-nitzschia brasiliana found in Malaysian waters, 30 strains of P. brasiliana were established into clonal culture since May 2008. The ultrastructure of these strains was examined for confirmation of species identification. The genetic marker, internal transcribed spacer (ITS) of nuclear-encoded ribosomal DNA was used to examine the genetic diversity of P. brasiliana isolated from different geographical localities. The ITS sequences of P. brasiliana were highly conserved in their secondary structures, with five helices in the first internal transcribe spacer (ITS1) and four universal helices in the second internal transcribe spacer (ITS2) with a pseudo-helix. No compensatory base change was observed among the strains examined. Genetic divergences among the Malaysian strains ranged from 0.07 to 0.54%. The present study revealed a high genetic homogeneity of Malaysian P. brasiliana strains.

Forty-eight isolates of Pseudo-nitzschia species were established from the Miri coast of Sarawak (Malaysian Borneo) and underwent TEM observation and molecular characterization. Ten species were found: P. abrensis, P. batesiana, P. fukuyoi, P. kodamae, P. lundholmiae, P. multistriata, P. pungens, P. subfraudulenta, as well as two additional new morphotypes, herein designated as P. bipertita sp. nov. and P. limii sp. nov. This is the first report of P. abrensis, P. batesiana, P. kodamae, P. fukuyoi, and P. lundholmiae in coastal waters of Malaysian Borneo. Pseudo-nitzschia bipertita differs from its congeners by the number of sectors that divide the poroids, densities of band striae, and its cingular band structure. Pseudo-nitzschia limii, a pseudo-cryptic species in the P. pseudodelicatissima complex sensu lato, is distinct by having wider proximal and distal mantles, a higher number of striae, and greater poroid height in the striae of the valvocopula. The species were further supported by the phylogenetic reconstructions of the nuclear-encoded large subunit ribosomal gene and the second internal transcribed spacer. Phylogenetically, P. bipertita clustered with its sister taxa (P. subpacifica + P. heimii); P. limii appears as a sister taxon to P. kodamae and P. hasleana in the ITS2 tree. Pairwise comparison of ITS2 transcripts with its closest relatives revealed the presence of both hemi- and compensatory base changes. Toxicity analysis showed detectable levels of domoic acid in P. abrensis, P. batesiana, P. lundholmiae, and P. subfraudulenta, but both new species tested below the detection limit.

A new species of Pseudo-nitzschia (Bacillariophyceae) is described from plankton samples collected from Port Dickson (Malacca Strait, Malaysia) and Manzanillo Bay (Colima, Mexico). The species possesses a distinctive falcate cell valve, from which they form sickle-like colonies in both environmental samples and cultured strains. Detailed observation of frustules under TEM revealed ultrastructure that closely resembles P. decipiens, yet the new species differs by the valve shape and greater ranges of striae and poroid densities. The species is readily distinguished from the curve-shaped P. subcurvata by the presence of a central interspace. The morphological distinction is further supported by phylogenetic discrimination. We sequenced and analyzed the nuclear ribosomal RNA genes in the LSU and the second internal transcribed spacer, including its secondary structure, to infer the phylogenetic relationship of the new species with its closest relatives. The results revealed a distinct lineage of the new species, forming a sister cluster with its related species, P. decipiens and P. galaxiae, but not with P. subcurvata. We examined the domoic acid (DA) production of five cultured strains from Malaysia by Liquid chromatography-mass spectrometry (LC-MS), but they showed no detectable DA. Here, we present the taxonomic description of the vegetative cells, document the sexual reproduction, and detail the molecular phylogenetics of Pseudo-nitzschia sabit sp. nov.

Domoic acid (DA) contamination in the thorny oyster Spondylus versicolor Schreibers 1793 was discovered in 2005, in Nha Phu Bay, Khanh Hoa Province, Vietnam. Concurrently, DA was detected in the net-plankton samples. The causative organism responsible for the DA was not detected then. In 2006, DA in S. versicolor (maximum of 43.6 µg·g -1 ) and in net-plankton samples (maximum of 0.78 ng·L -1 ) recurred, suggesting the existence of DA producers in the bay. When DA in S. versicolor again increased in 2007, a net-plankton sample was collected, and cultures of Pseudo-nitzschia species were established for DA analysis and species identification. Eight out of eleven cultured isolates of Pseudo-nitzschia spp. showed DA production (111–244 ng·mL -1 ), as confirmed by liquid chromatography-tandem mass spectroscopy. The toxic isolates examined by transmission electron microscopy shared identical morphological characteristics: a single row of poroids, hymens divided into 2–6 sectors, and mantles 1–2 poroids high. They resembled Pseudonitzschia caciantha Lundholm, Moestrup & Hasle, 2003 thus we designated it as P. cf. caciantha . Our results indicated that P. cf. caciantha most likely contributed to the DA contamination in S. versicolor in Nha Phu Bay. This is the first report of DA production by P. cf. caciantha anywhere in the world.

Field sampling was undertaken to investigate the occurrence of Pseudo-nitzschia Peragallo species in eight locations along the coast of Malaysian Borneo. A total of 108 strains of Pseudo-nitzschia species were isolated, and their morphology examined with SEM and TEM. Additionally, molecular data from nuclear-encoded partial LSU rDNA, and ITS regions, were characterized. A total of five species were confidently identified based on a combination of distinct morphological characteristics and supporting molecular evidence: P. brasiliana Lundholm, Hasle & Fryxell, P. cuspidata (Hasle) Hasle, P. dolorosa Lundholm & Moestrup, P. micropora Priisholm, Moestrup & Lundholm, and P. pungens (Grunow) Hasle var. pungens. However, one morphotype from Sarawak, while somewhat similar to P. caciantha, showed significant morphological distinction from this and any other of the currently described species. Most notably this morphotype possessed a characteristic pore arrangement in the poroids, with the fine pores in each perforation sector arranged in circles. Pair-wise sequence comparison of the LSU rDNA between this unidentified morphotype and P. caciantha Lundholm, Moestrup & Hasle, revealed 2.7% genetic divergence. Phylogenetic analyses strongly supported the monophyly of the morphotype. Based upon these supporting data it is here described as a new species, Pseudo-nitzschia circumpora sp. nov. A key to the six species of Pseudo-nitzschia from Malaysian Borneo is presented. Molecular signatures for all species were established based on structural comparisons of ITS2 rRNA transcripts.